CHARACTERIZATION OF DIVERSE NONCODING RNAS IN THERAPEUTICS, BIOTECHNOLOGY AND CHROMATIN BIOLOGY

Author

Ramadevi Chilamkurthy, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2024

Degree Name

Doctor of Philosophy

Department

Molecular Biology, Microbiology and Biochemistry

First Advisor

Gagnon, Keith

Abstract

This study encompasses exploration of diverse noncoding RNAs (ncRNAs) across therapeutics, biotechnology, and cell biology, presented through three distinct yet interconnected projects. The first project delves into the 3D organization of the mammalian genome, particularly focusing on "mitotic bookmarking" and the potential role of RNA in maintaining genomic integrity and cellular memory during cell division. By employing advanced chromatin conformation capture techniques, notably the iMARGI method, this study generated a comprehensive RNA-DNA interactome across cell cycle phases, revealing significant cell type-specific differences in RNA-DNA interactions. These findings highlight the dynamic interplay between RNA and DNA and will contribute to a deeper understanding of genomic regulation and cellular memory. The second project investigates the conformational dynamics and target sequence influence on the catalytic activity of single versus dual RNA-guided CRISPR-Cas9 systems. Contrary to conventional wisdom, our research found that dual guide RNAs (dgRNAs) can perform as well as, or better than, single guide RNAs (sgRNAs) in genome editing efficiency. This discovery was supported by molecular dynamics simulations, revealing that dgRNAs and sgRNAs confer alternative structural dynamics to Cas9, impacting its target sequence preferences and catalytic activity. This study thus provides crucial insights into the structural underpinnings of CRISPR-Cas9 efficiency and paves the way for optimizing gene editing approaches. The third project focuses on the challenges of cloning and sequence validation of repetitive and high GC-content shRNAs, often used in gene knockdown and therapeutic applications. We present improved methods for efficient cloning of shRNAs targeting disease-associated repeat expansions into lentivectors, enhanced by design and preparation techniques, recombination-based cloning, and comprehensive sequencing-based validation. These advancements in shRNA technology underscore the importance of RNA-based tools in therapeutic development and gene research. In summary, this dissertation provides a comprehensive examination of the functional versatility of ncRNAs, from their role in chromatin organization and gene regulation to their applications in biotechnological innovations and therapeutic strategies. The findings collectively underscore the significance of ncRNAs in advancing our understanding of complex biological systems and their potential in developing innovative solutions to address diverse biomedical challenges.